Arrangement and method for performing chromatography

ABSTRACT

An arrangement for performing chromatography with a mobile phase and a chromatographic bed having a stationary phase is provided. The arrangement includes a support assembly configured to support the stationary phase. The support assembly has a sealed cavity configured so that when the support assembly is supporting the stationary phase a portion of the chromatographic bed and the stationary phase extends into the sealed cavity. The arrangement further includes a bladder having a void defined therein. The bladder is positioned within the sealed cavity. The arrangement also includes a fluid positioned within the void of the bladder so that the bladder is urged toward the stationary phase within the sealed cavity. The arrangement further includes an anode positioned in contact with the mobile phase. The arrangement also includes a cathode positioned in contact with the stationary phase, wherein creating an electrical potential between the anode and the cathode causes the mobile phase to be advanced through the chromatographic bed positioned within the sealed cavity. An associated method is also provided.

BACKGROUND OF THE INVENTION

The present invention generally relates to an arrangement and method forperforming chromatography. The present invention particularly relates toa an arrangement and method for performing chromatography utilizingelectroosmotic flow of a mobile phase.

Multiple techniques have been developed which enable the separation ofcomplex mixtures into their components. Chromatography is one suchtechnique. Chromatography can be described as a separation process basedon differences in the rate at which the components of a mixture movethrough a chromatographic bed under the influence of a mobile phasewhich moves relative to a chromatographic bed. The chromatographic bedwill typically include a plurality of porous or microporous particles,such as bonded C₁₈ silica, wherein the collective surface of theseparticles make up the stationary phase. Several types of chromatographysystems have the chromatographic bed packed into the interior of acolumn. Alternatively, the chromatographic bed can be dispersed on aglass plate. An example of a chromatography system that utilizes thechromatographic bed packed into a column is High Performance LiquidChromatography (hereinafter referred to as HPLC). An example of achromatography system that utilizes the chromatographic bed dispersed ona glass plate is Thin Layer Chromatography (hereinafter referred to asTLC) or Overpressurized Layer Chromatography (hereinafter referred to asOPLC).

As previously mentioned HPLC involves packing the chromatographic bedwithin the interior of a column. The mobile phase is then pumped throughthe column (and thus through the chromatographic bed) at a very highpressure. A sample is then introduced into the chromatographic systemand is pumped through the chromatgraphic bed. As the sample is pumpedthrough the chromatographic bed the components of the sample arepartitioned between the mobile phase and the stationary phase based upontheir differing physical and chemical characteristics. For example, thecomponents of the mixture can be partitioned between the mobile andstationary phases based upon their polarity, charge, and size. Since thecomponents of a mixture will typically differ based upon their polarity,charge, and size they can be separated from each other by advancing themthrough the chromatographic bed.

HPLC is a very useful chromatographic technique, however it does sufferfrom several disadvantages. For example, (i) HPLC system can onlyseparate one mixture at a time, (ii) HPLC systems require special pumpsand inlet devices to respectively generate and accommodate the highpressures required to perform HPLC, (iii) HPLC columns must beconstructed from mechanically strong materials which limits the use ofglass columns that are particularly useful for handling many biologicalsamples, (iv) HPLC systems designed for preparative chromatographytechniques are very expensive, and (v) detector dead volumes must bekeep extremely small (several microliters) in order to avoid additionaland spreading.

With respect to TLC, the chromatographic bed is a layer (0.1-0.5 mmthick) of a sorbent material spread uniformly over the surface of aglass or plastic plate. The mixture to be separated is applied to thechromatographic bed with a micropipette and dried. The TLC plate is thenplaced in a chamber so that a small portion of the stationary phase isin contact with a mobile phase. The TLC plate is developed by allowingthe mobile phase to ascend up the plate by capillary action. The basisfor the separation of the mixture into its respective components is thesame as discussed above with respect to HPLC, i.e. the components areseparated due to their different partitioning between the stationary andmobile phases. This in turn is based upon the differing polarity,charge, and size characteristics of each of the components of themixture to be separated.

However, like HPLC, TLC also suffers from several significantdisadvantages. In particular, the separation efficiency by TLC islimited by the inadequate mobile phase flow under capillary action. Thiscapillary-induced mobile phase flow is neither fast enough nor constantthroughout the chromatographic run, and both of these drawbacks tend todecrease the separation efficiency of TLC substantially. Moreover, therelatively slow movement of the mobile phase results in rather longdevelopment times.

OPLC attempts to overcome the aforementioned difficulties associatedwith TLC. This technique forces the mobile phase through thechromatographic bed disposed on the plate by applying high pressure tothe mobile phase. This results in a flow rate that can be controlled andremains constant throughout the development of the plate. A consequenceof the constant flow rate is that the number of theoretical platesencountered by a solute will increase linearly with increasing migrationdistance. In addition, the total time of an analysis is substantiallydecreased because the mobile phase flows faster.

OPLC also suffers from significant drawbacks. In particular, the flow ofthe mobile phase in OPLC systems is laminar. Laminar flow profile orparabolic flow profile means that throughout the cross-sectional area ofthe mobile phase within a channel between particles the center portionof the liquid of the mobile phase flows faster than the liquid close tothe wall of the channel. The laminar flow profile of OPLC systemsresults in migration characteristics of the mobile phase being sensitiveto the particle size and the particle size distribution of thestationary phase. Having the migration characteristics of the mobilephase sensitive to the particle size and the particle size distributionof the stationary phase can decrease the separation efficiency of OPLC.

What is needed therefore is a chromatographic arrangement and methodwhich overcomes one or more of the aforementioned problems.

SUMMARY OF THE INVENTION

In accordance with one embodiment of the present invention, there isprovided an arrangement for performing chromatography with a mobilephase and a chromatographic bed having a stationary phase. Thearrangement includes a support assembly configured to support thechromatographic bed and the stationary phase. The support assembly has asealed cavity configured so that when the support assembly is supportingthe stationary phase a portion of the chromatographic bed and thestationary phase extends into the sealed cavity. The support assemblyalso includes a first electrode positioned in contact with the mobilephase. The arrangement further includes a second electrode positioned incontact with the stationary phase, wherein creating an electricalpotential between the first electrode and the second electrode causesthe mobile phase to be advanced through the chromatographic bedpositioned within the sealed cavity.

Pursuant to another embodiment of the present invention there isprovided a method of performing chromatography with a chromatographicbed having a stationary phase. The method includes the steps of (a)positioning the chromatographic bed and the stationary phase within asealed cavity, (b) placing the stationary phase in contact with a liquidmobile phase while the stationary phase is positioned within the sealedcavity, (c) advancing a fluid into the sealed cavity so that pressurewithin the sealed cavity is greater than the pressure outside of thesealed cavity, (d) placing a first electrode in contact with the liquidmobile phase, (e) placing a second electrode in contact with thestationary phase, and (f) creating an electrical potential between thefirst and second electrode so as to cause the liquid mobile phase to beadvanced through the chromatographic bed positioned within the sealedcavity.

According to yet another embodiment of the present invention, there isprovided an arrangement for performing chromatography with a mobilephase and a chromatographic bed having a stationary phase. Thearrangement includes a support assembly configured to support thechromatographic bed and the stationary phase. The support assembly has asealed cavity configured so that when the support assembly is supportingthe stationary phase a portion of the chromatographic bed and thestationary phase extends into the sealed cavity. The arrangement alsoincludes a fluid positioned within the sealed cavity so that (i) thefluid is placed in a heat exchange relationship with the stationaryphase and (ii) pressure within the sealed cavity is greater thanpressure outside of the sealed cavity. The arrangement further includes(i) an anode positioned in contact with the mobile phase and (ii) acathode positioned in contact with the stationary phase, whereincreating an electrical potential between the anode and the cathodecauses the mobile phase to be advanced through the chromatographic bedpositioned within the sealed cavity.

It is therefore an object of the present invention to provide a new anduseful an arrangement and method for performing chromatography.

It is another object of the present invention to provide an improvedarrangement and method for performing chromatography.

It is still another object of the present invention to provide anarrangement and method for performing chromatography which cansimultaneously separate multiple mixtures.

It is moreover an object of the present invention to provide anarrangement and method for performing chromatography which canefficiently separate the components of a mixture.

It is yet another object of the present invention to provide anarrangement and method for performing chromatography which does notrequire relatively expensive pumps and inlet devices.

It is still another object of the present invention to provide anarrangement and method for performing chromatography which requiresrelatively short development times.

It is yet another objective of the present invention to have the mobilephase at optimum velocity for maximum chromatographic efficiency.

The above and other objects, features, and advantages of the presentinvention will become apparent from the following description andattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary schematic representation of a chromatographyarrangement which incorporates certain features of the present inventiontherein;

FIG. 2A is a schematic representation of a plug flow profile of themobile phase obtained with the chromatography arrangements of thepresent invention;

FIG. 2B is a schematic representation of a laminar flow profile(parabolic profile) of the mobile phase obtained with chromatographyarrangements such as OPLC;

FIG. 3 is perspective view of another chromatography arrangement whichincorporates certain features of the present invention therein, notethat a housing is shown in phantom for clarity of description;

FIG. 4 is partially schematic front elevational view of anotherchromatography arrangement which incorporates certain features of thepresent invention therein;

FIG. 5 is a partially schematic side elevational view of the arrangementshown in FIG. 4;

FIG. 6 is an end elevational view of the chromatography arrangementshown in FIG. 4 as viewed in the direction of line 6—6;

FIG. 7 is an enlarged cross sectional fragmentary view of an upperportion of the chromatography arrangement of FIG. 5 showing the wick andcathode configuration (note that the elements of the cathode areenlarged for clarity of description);

FIG. 8 is a cross sectional view of a chromatography arrangement similarto the one shown in FIG. 5, but showing a bladder disposed within thesealed cavity of the container;

FIG. 9A depicts a chromatogram obtained by the chromatographyarrangement shown in FIG. 3 utilizing an 80% aqueous ethanol (v/v) (8parts ethanol and 2 parts water) mobile phase containing 1 mmol TAPSbuffer with an applied electrical potential of 2000 volts;

FIG. 9B depicts a chromatogram obtained by conventional TLC utilizing an80% aqueous ethanol (v/v) mobile phase containing 1 mmol TAPS buffer;

FIG. 10 depicts a graph showing the average velocity of the highest (o)and lowest (x) dye components of a mixture obtained by the appliedpotential utilizing the chromatography arrangement shown in FIG. 3 withan 80% aqueous ethanol (v/v) mobile phase containing 1 mmol TAPS buffer;and

FIG. 11 depicts a graph showing distance traveled and elapsed time for aseparation obtained by the chromatography arrangement shown in FIG. 3utilizing an 80% aqueous ethanol (v/v) mobile phase containing 1 mmolTAPS buffer with an applied electrical potential of 2000 volts.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

While the invention is susceptible to various modifications andalternative forms, a specific embodiment thereof has been shown by wayof example in the drawings and will herein be described in detail. Itshould be understood, however, that there is no intent to limit theinvention to the particular form disclosed, but on the contrary, theintention is to cover all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theappended claims.

Referring to FIG. 1 there is shown a chromatography arrangement 10 whichincorporates certain features of the present invention therein.Arrangement 10 includes a thin layer chromatography plate 12(hereinafter referred to as plate 12), an electrical power source 40, afirst electrode 28 such as an anode, and a second electrode 30 such as acathode. Arrangement 10 also includes a mobile phase 24 and a pair ofelectrical wires 42 and 44. Hereinafter first electrode 28 will bereferred to as anode 28 and second electrode 30 will be referred tocathode 30. However, it should be understood that a chromatographyarrangement where first electrode 28 is a cathode and second electrode30 is an anode is also contemplated whereby the chemistry of stationarylayer 16 is appropriately altered to accommodate the above describedchange in the positions of first electrode 28 and second electrode 30.

Plate 12 includes support plate 14 such as a glass or plastic plate.Plate 12 also includes a chromatographic bed 16 disposed and adheredonto a surface of support plate 14 in a well known manner. Plate 12 alsoincludes a cathode portion 170, an anode portion 172, a sample area 22,an end 18, and an end 20. An example of a chromatographic bed 16 whichcan be used in the present invention is silica gel having a uniformparticle size. In particular, the particle size of the silica gel can befrom about 2 micrometers to about 10 micrometers. However, it should beunderstood that the size distribution of the particles should be asnarrow as possible. Moreover, the thickness of the chromatographic bed16 disposed onto the surface of support plate 14 can be from about 0.1millimeters to about 0.25 millimeters thick. However, thicker stationaryphases 16 can be used when performing preparative chromatography. Plates12 which satisfy the aforementioned criteria and can be used in thepresent invention are commercially available as catalogue number 15389(RP-18 F_(254s) plates; note that chromatographic bed of RP-18 is madeup of modified silica) from Merck, which is located in Darmstadt,Germany. Preferably, the aforementioned commercially available plates 12are conditioned at 120° C. for 20 minutes prior to use in the presentinvention.

Mobile phase 24 is preferably a liquid. An example of a mobile phasewhich can be utilized in the present invention is 80% ethanol/water(v/v) with a final {3-[tris(hydroxymethyl amino]-1-propanesulfonic acid}(herein after referred to as TAPS) buffer concentration of about 0.5mmol to about 2.5 mmol. TAPS is commercially available as cataloguenumber 21,993-2 from the Aldrich Chemical Company, which is located inMilwaukee, Wis.

All of plate 12 is pre-wetted, except sample area 22, by dipping plate12 in an aqueous solution whose buffer concentration matched that of themobile phase 24. Excess liquid is removed by blotting chromatographicbed 16 of plate 12 on a paper towel. A sample mixture to be separated isspotted onto sample area 22 with a micropipette (not shown), amicroliter syringe (not shown), or any other appropriate spottingdevices prior to pre-wetting the plate 12. Note that the volume of thesample mixture was less that 0.1 microliters. Preferably, the initialspot containing the sample mixture placed onto sample area 22 of plate12 should be kept as small as possible. In addition, the initial spot ispreferably positioned to within 2 millimeters of the pre-wetted portionof plate 12. Note that spot 34, representing the initial spot of thesample mixture to be separated, is shown enlarged for clarity ofdescription.

The plate 12 is positioned relative to the mobile phase 24 such that (i)end 20 of plate 12 is located below the surface 26 of mobile phase 24and (ii) sample area 22 with spot 34 disposed thereon, is located abovethe surface 26 of mobile phase 24. It should be understood that a tankcan be used to contain mobile phase 24 as shown in FIG. 3.

Anode 28 is electrically coupled to power source 40 via electrical wire42. In addition, cathode 30 is electrically coupled to power source 40via electrical wire 44. Anode 28 is placed in contact with mobile phase24. Cathode 30 is placed into contact with cathode portion 170 of plate12. Preferably, cathode 30 is urged into direct contact withchromatographic bed 16 with a clamping mechanism, e.g. an electricallynon-conducting clamp. Once cathode 30 and anode 28 are positioned asdescribed above and electrically coupled to power source 40 anelectrical potential is created between cathode 30 and anode 28 withpower source 40. It should be understood that the electrical potentialshould be created between cathode 30 and anode 28 about 10 seconds toabout 30 seconds after end 20 of plate 12 is located below the surface26 of mobile phase 24.

The magnitude of the electrical potential created with power source 40is limited by the amount of current the power source 40 can tolerate,and by the ohmic heating which can cause plate 12 to dry out during thedevelopment thereof. For example, in the present invention theelectrical potential generated by power source 40 can range from about500 V to about 2500 V. A power source which can be used in the presentinvention for generating the aforementioned electrical potentials iscommercially available from the Spellman High Voltage ElectronicsCompany, located in Plainview, N.Y., as model SL30P600 or model numberSL30N600.

When the aforementioned electrical potential is generated between anode28 and cathode 30 cations present in the mobile phase 24 are attractedto the negatively charged cathode 30. This migration of cations towardthe cathode causes mobile phase 24 to be advanced through thechromatographic bed 16 in the direction indicated by arrow 32, i.e.toward cathode 30. The process of advancing mobile phase 24 throughchromatographic bed 16 by placing a first electrode 28 in contact withmobile phase 24 and a second electrode 30 in contact withchromatographic bed 16 and then generating an electrical potentialbetween first electrode 28 and second electrode 30 will hereinafter bereferred to as electroosmosis.

As previously discussed, as mobile phase 24 is advanced toward cathode30 the components of the mixture contained within initial spot 34partition between mobile phase 24 and chromatographic bed 16 based upontheir differing physical and chemical characteristics. Since thecomponents of mixture contained within initial spot 34 will typicallydiffer based upon their polarity, charge, and size they are separatedfrom each other as plate 12 is developed, i.e. as the front 176 ofmobile phase 24 is advanced through chromatographic bed 16 away fromanode position 172 and toward cathode position 170.

An exemplary separation is depicted in FIG. 1. In particular, themixture initially disposed onto sample area 22 of plate 12 as spot 34 isdepicted as containing two components, i.e. spot 104 and spot 106. Asshown in FIG. 1, utilizing chromatography arrangement 10 as describedabove results in these two components being separated from each otheralong the longitudinal axis of plate 12. Once separated, spots 104 and106 can be detected or visualized with various well known techniques.For example, after development and drying, spots 104 and 106 could bevisualized by scanning plate 12 with a dual wavelength flying-spotscanner at λ=254 nm in the reflectance mode. One such scanner which canbe used in the present invention is commercially available from theShimadzu Corporation, located in Kyoto, Japan as model number CS900Udual wavelength flying-spot scanner.

It should be appreciated that utilizing electroosmosis to advance mobilephase 24 through chromatographic bed 16 has several advantages overadvancing a mobile phase through a chromatographic bed with pressure orcapillary action. In particular, as shown in FIG. 2A, utilizingelectroosmosis to advance mobile phase 24 through chromatographic bed 16in the direction of arrow 150 results in mobile phase 24 having a plugflow profile 183. Having a plug flow profile results in the crosssectional velocity of the flow of mobile phase 24 being constant. Thisresults in a reduction in transaxial zone broadening, whichsubstantially increases the separation efficiency of chromatographyarrangement 10 as compared to other chromatography arrangements whichutilize pressure or capillary action to advance the mobile phase throughthe stationary phase. Specifically, chromatography arrangements whichdepend upon pressure or capillary action to advance the mobile phasethrough the chromatographic bed have laminar mobile phase flow profiles(i.e. parabolic flow profiles).

In particular, in FIG. 2B there is shown a flow profile 177 of a mobilephase 179 being advanced through a chromatographic bed 181 in thedirection indicated by arrow 156 with pressure. As previously mentioned,advancing a mobile phase through a chromatographic bed with pressureresults in a laminar flow profile. In other words, the center portion ofthe liquid of mobile phase 179 flows faster than the liquid close to thesurface as mobile phase 179 is advanced through chromatographic bed 181.This laminar flow profile increases the transaxial contributions to zonebroadening which substantially decreases the separation efficiency ofsuch pressure driven chromatography arrangements. Moreover, having apressure driven mobile phase results in the migration characteristics ofthe mobile phase being sensitive to (i) the particle size and (ii) theparticle size distribution of the stationary phase. Having the migrationcharacteristics of the mobile phase sensitive to the aforementionedparameters also decreases the separation efficiency of such pressure orcapillary action driven mobile phase chromatography arrangements.

Furthermore, utilizing electroosmosis to advance mobile phase 24 throughchromatographic bed 16 has several additional advantages over advancinga mobile phase through a chromatographic bed with pressure or capillaryaction. These advantages include the ability to achieve optimum andconstant linear velocity of the mobile phase, and an increased totalnumber of theoretical plates available for separation. Additionally,with electroosmosis the length of the chromatographic bed (e.g. thelength of plate 12) will no longer be a limiting factor in gaininggreater efficiency because the decrease in linear velocity with distancetraveled will no longer be an issue as in capillary mediatedchromatography arrangements. In other words their is no theoreticallimit to the length the chromatographic bed can be used in thisarrangement. Furthermore, the flow rate of the mobile phase inelectroosmotic systems is independent of the particle size and packinguniformity of the chromatographic bed which facilitates a greaterseparation efficiency.

Referring now to FIG. 3, there is shown a chromatography arrangement 37which is similar to arrangement 10. Arrangement 37 functions insubstantially the same way, and has the same advantages, as discussedabove in reference to arrangement 10. In particular, it should beunderstood that arrangement 37 also drives the mobile phase 24 throughthe chromatographic bed 16 of plate 12 utilizing an electrical potentialgenerated between first electrode 28 and second electrode 30. However,arrangement 37 includes a support assembly 36 for supporting plate 12,and therefore chromatographic bed 16. Arrangement 37 also includes anenclosure 43 which surrounds support assembly 36 and plate 12 when plate12 is being developed. Enclosure 43 includes a door (not shown)operatively coupled to a safety switch (not shown) which disables powersource 40 when the door is opened. In addition, arrangement 37 differsfrom arrangement 10 in that the cathode 30 of arrangement 37 includes apiece of platinum foil 52 (about 0.8×2 cm) spot welded to a platinumwire 50. Note that the platinum wire 50 shown in FIG. 3 is enlarged forclarity of description. Moreover, arrangement 37 also includes a wick 75for absorbing the liquid of mobile phase 24. The absorptive capacity ofwick 75 can be augmented by a porous bag filled with an absorptivematerial and then placing the porous bag in intimate contact with wick75.

Support assembly 36 includes a frame 38, a plate 39, a plate 41 and atank 73. Support assembly 36 also includes brackets 58, 59, 61, and 67.The aforementioned elements can be made of delrin or Plexiglas. Notethat all of the parts that may come into contact with the mobile phaseor its vapor should be made out of delrin. Also note that each ofbrackets 58, 59, 61, and 67 include an elongated slot 60 definedtherein. Support assembly 36 also includes clamping member 54 and aclamping member 56. Clamping member 54 has a first notch 57 and a secondnotch 185 defined therein. In a similar manner, clamping member 56 has afirst notch 55 and a second notch 187 defined therein.

Plates 39 and 41 are secured to frame 38 as shown in FIG. 3. Bracket 58is secured to plate 39 with a screw 62 that extends through elongatedslot 60 and a hole (not shown) defined in plate 39. A nut 63 is thenmeshingly engaged with screw 62 to ensure that bracket 58 remainssecured to plate 39 (a washer can also be interposed between nut 63 andbracket 58 if necessary). It should be appreciated that securing bracket58 to plate 39 in the above described manner allows bracket 58 to moverelative to plate 39 in the direction indicated by arrows 71 and 72.However, bracket 58 can be locked into position relative to plate 39 bytightening nut 63. Bracket 59 is secured to plate 39 in a substantiallyidentical manner as that described for bracket 58. Thus, bracket 59 canalso be moved relative to plate 39 in the directions indicated by arrows71 and 72 or locked into position relative to plate 39 by tighteningnut. Brackets 61 and 67 are secured to plate 41 in a substantiallyidentical manner as that described for bracket 58. In addition, brackets61 and 67 function in a substantially identical manner as that describedfor bracket 58.

Clamping member 54 is secured to brackets 58 and 59 so clamping member54 can also move relative to plate 39 in the directions indicated byarrows 71 and 72. In a similar manner clamping member 56 is secured tobrackets 61 and 67 such that clamping member 56 can move relative toplate 41 in the directions indicated by arrows 71 and 72.

Each clamping member 54 and 56 has a holding mechanism 64 and 70,respectively, attached thereto. Holding mechanism 64 includes a screw 65extending through clamping member 54. An intermediate member 68 is thendisposed over screw 65. A nut 66 is then meshingly engaged with screw 65so that intermediate member 68 is interposed between nut 66 and clampingmember 54. Holding member 70 is attached clamping member 56 in asubstantially identical manner as that described for holding member 64.

During use of arrangement 37 plate 12 is pre-wetted as described above.The sample to be separated is then spotted on sample area 22 of plate12. Plate 12 is then positioned relative to clamp member 54 and clampmember 56 such that (i) one edge of plate 12 is located within secondnotch 185 of clamping member 54 and (ii) the opposite edge of plate 12is located within second notch 187 of clamping member 56. Plate 12further positioned such that end 20 is located below the surface 26 ofmobile phase 24. However, sample area 22 should be located above surface26 of mobile phase 24. Once positioned as described above, clampingmembers 54 and 56 are gently urged together. Nuts 63,189, 191, and 193are then tightened to lock clamping members 54 and 56 in their place.

Platinum foil 52 of cathode 30 is placed into contact with cathodeportion 170 of plate 12. Wire 44 is electrically coupled to platinumwire 50 and power source 40. Note that arrangement 37 incorporates asecurement mechanism 46 attached to frame 38 for supporting wire 44. Aswith arrangement 10, anode 28 is placed in contact with mobile phase 24and electrically coupled to power source 40 with wire 42 so that mobilephase 24 is advanced up plate 12 in the direction indicated by arrow 86.

As more clearly shown in FIG. 7, wick 75 is preferably a piece of filterpaper. The filter paper is folded along an edge thereof so that a 1-2 mmlip is created. The lip is then positioned underneath a bottom edge ofplatinum foil 52 so that the lip is interposed between platinum foil 52and chromatographic bed 16 of plate 12. The remaining portion of wick 75extends out in front of cathode 30 in an upwardly direction. The lengthof wick 75 can be increased so that wick 75 extends upwardly for asubstantial distance above the end of plate 12. Doing so enhances theabsorptive capacity of wick 75.

A glass plate 48 is disposed over chromatographic bed 16 of plate 12such that opposing edges of glass plate 48 are located within notches 55and 57 of clamping members 56 and 54. In addition, a number of spacers(not shown) are interposed between glass plate 48 and plate 12 such thatan air gap 194 about ⅛ of an inch wide is created between glass plate 48and plate 12. Both intermediate members 68 and 69 are disposed aroundtheir respective screws (e.g. screw 65). The nuts 66 and 74 of holdingmembers 64 and 70 are tightened such that intermediate members 68 and 69are urged toward glass plate 48. Urging that intermediate members 68 and69 toward glass plate 48 holds plate 12 and glass plate 48 interposedbetween clamping members 54 and 56. Moreover, a small piece of rubber(not shown) is interposed between glass plate 48 and platinum foil 52 sothat the rubber piece urges platinum foil 52 against chromatographic bed16 when intermediate members 68 and 69 are urged against glass plate 48.

A dye mixture was separated utilizing arrangement 37. The dye mixtureseparated is commercially available from Analtech, located in Newark,N.J., as catalogue number 30-04 (Test Mixture IV). The dye mixture wasspotted onto sample area 22 of plate 12. Plate 12 was then prepared asdescribed above and placed into support assembly 36 as previouslydescribed for development.

The dye mixture was separated using an electrical potential of 2000 Vand a 1.0 mmol TAPS buffer concentration on a plate 12 having thedimensions of 2.5×10.0 cm. The same separation using conventional TLCchromatography was also performed. The development of each plate wasterminated when the mobile phase reached 5.5 cm from the bottom edge ofthe plate. The plate 12 utilizing arrangement 37 developed in 18 min. ascompared to 37.5 min. for the plate developed with conventional TLCchromatography.

Table 1 sets forth the width of the peaks at half height (w_(0.5)), themigration distance (MD), and the number of theoretical plates (N)obtained in each separation. The peaks obtained utilizing arrangement 37(electroosmosis) are more narrow (0.17 to 0.18 cm) as compared to theconventional TLC chromatography arrangement (0.18 to 0.38 cm). Moreover,the number of theoretical plates utilized was higher by a factor of 2.5to 4.6 in arrangement 37 as compared to the conventional TLCchromatography arrangement for the same distance traveled. Thechromatogram obtained for arrangement 37 and the conventional TLCarrangement is shown in FIGS. 9A and 9B, respectively.

TABLE 1 Conventional TLC Electroosmosis Conventional Solute MD (cm)w_(0.5) (cm) N MD (cm) w_(0.5) (cm) N 1 1.34 0.18 325 0.60 0.18 60 22.19 0.17 964 1.22 0.25 132 3 2.61 0.17 1370 1.67 0.27 218 4 3.22 0.181715 2.34 0.33 274 5 3.79 0.17 2888 3.08 0.38 374 6 4.49 0.18 3647 4.510.29 1331

In addition, the elapsed time and distance traveled of the highestmigrating dye component in arrangement 37 is shown in Table 2. Theseresults are graphically shown in FIG. 11.

TABLE 2 Time (min) Distance traveled (cm) 0.90 1.0 3.68 2.0 7.31 3.011.92 4.0 18.00 4.5

The dye mixture was separated using 1.0 mmol TAPS buffer concentrationand an applied potential ranging from 500 V to 2500 V in steps of 500 V.Plates 12 were cut into 2×10 cm sections and then used. However, muchlarger plates can be utilized. The separation was terminated when thehighest migrating dye component traveled 5 cm from the bottom edge ofthe plate 12. Table 3 lists the applied potential, time required for theseparation, and the average velocity of the highest and lowest migratingdye components. FIG. 10 is a plot of the average velocity vs. appliedpotential. The average velocity increases with increasing appliedpotential. The highest potential applied is limited by the amount ofcurrent the power supply can tolerate, and by ohmic heating causingplate 12 to dry. The plates 12 used in the subject voltage study wereslightly more narrow than those previously used (2 cm as compared to 2.5cm). This allows a slightly higher potential to be applied without thecurrent exceeding the limitations of the power source.

TABLE 3 Applied Total time of Average velocity Average velocitypotential development of highest spot of lowest spot (kV) (min) (cm/min)(cm/min) 0.5 37.82 0.105 0.024 1.0 26.10 0.151 0.040 1.5 20.25 0.1940.057 2.0 14.33 0.267 0.086 2.5 9.95 0.402 0.154

Still referring to FIG. 3, there is shown a housing 76 which can be usedin conjunction with frame 38 of assembly 37. Housing 76 has an interiorchamber 78 defined therein. Housing 76 is also equipped with an entranceport 80 and an exit port 82. Both entrance port 80 and exit port 82 arein fluid communication with interior chamber 78.

When housing 76 is used in conjunction with arrangement 37, housing 76is positioned relative to frame 38 so that it is located adjacent to aback surface of plate 12. In particular, housing 76 is located adjacentto the back surface of plate 12 so that a space 84 is defined betweenhousing 76 and the back surface of plate 12 (about {fraction (1/16)} ofan inch separation between housing 76 and plate 12). Entrance port 80 isthen placed in fluid communication with a pump (not shown) for advancinga cooling fluid 174 through interior cavity 78. Specifically, the pumpcirculates cooling fluid 174 through interior chamber 78 of housing 76via entrance port 80 and exit port 80 during the development of plate12.

Having housing 76 positioned relative to plate 12 in the aforementionedmanner is an advantage, in that cooling fluid 174 facilitates keepingthe temperature of plate 12 within an acceptable range during thedevelopment of plate 12. Specifically, cooling fluid prevents plate 12from becoming over heated during the development of plate 12 and thusenhances the separation efficiency of arrangement 37.

Now referring to FIGS. 4, 5, 6, and 7, there is shown a chromatographyarrangement 88 which is similar to arrangement 37. Arrangement 88functions in substantially the same way, and has the same advantages, asdiscussed above in reference to arrangement 37. In particular, it shouldbe understood that arrangement 88 also drives the mobile phase 24through the chromatographic bed 16 of plate 12 utilizing an electricalpotential generated between first electrode 28 and second electrode 30.However, support assembly 36 of arrangement 88 includes a container 90for enclosing a portion 166 of chromatographic bed in a sealed cavity100. Arrangement 88 also includes a temperature control unit 128, a pump122, a cooling unit 132, and a temperature sensor 126.

Container 90 includes a first member 92 and a second member 94. Firstmember 92 and second member 94 are preferably constructed from anon-electrically conducting ceramic material encased in polycarbonate(Lexan). Polycarbonate which can be utilized in the present invention toencase the ceramic material is commercially available from theMcMaster-Carr Supply Company located in Chicago, Ill.

First member 96 has a seat area 96 and a number of holes 102 (see FIG.6) defined therein. First member 92 is secured to frame 38 so that firstmember 92 can be held in an upright position as shown in FIGS. 4 and 5.Second member 94 has cut out portion 98 and a number of holes 102 (seeFIG. 6) defined therein. In addition, second member 94 has a channel 146defined therein as shown in FIG. 7. A gasket 112 is positioned withinchannel 146.

When using arrangement 88 to perform chromatography, plate 12 is firstpre-wetted as described above. In addition, edges 150 and 152 of plate12 are coated with a sealant 148 as shown in FIG. 4. The sealant 148 canbe an epoxy based resin which is chemically inert to mobile phase 24,but provides a relatively hard surface on which gasket 112 and membrane144 can rest. The sample to be separated is then spotted on sample area22 of plate 12.

Plate 12 is then positioned relative to first member 92 so that plate 12is located within seat area 96 (see FIG. 6). As more clearly shown inFIG. 7, a pliable membrane 144 is positioned in contact withchromatographic bed 16 so that chromatographic bed 16 is interposedbetween membrane 144 and support 14. Membrane 144 can be made out of anyappropriate material which has a low electrical conductivity and arelatively high thermal conductivity. For example, the membrane 144 usedin the present invention can be made out of a {fraction (1/16)} inchthick sheet of polytetrafluoroethylene (Teflon). In addition, membrane144 can include a sheet of mylar attached to the surface of the Teflonsheet which faces toward sealed cavity 100. Moreover, membrane 144 caninclude a sheet of Kel-F CTFE attached to the surface of the Teflonsheet which faces toward sealed cavity 100. Kel-F CTFE is commerciallyavailable from McMaster-Carr Supply Company, located in Chicago, Ill.Yet another material which can be incorporated into membrane 144 is AN90 aluminum nitride ceramic which is commercially available fromMarkeTech located in Port Townsend, Wash. However, as with mylar if AN90 aluminum nitride ceramic is incorporated into membrane 144 the Teflonsheet must contact the chromatographic bed. Furthermore, membrane 144can include any sandwich combination of the above mentioned materials aslong as the Kel-F CTFE or Teflon sheet is in contact with thechromatographic bed 16.

Once plate 12 is positioned within seat area 96, and membrane 144 isplaced into contact with chromatographic bed 16, second member 94 ispositioned relative to first member 92 so that the holes 102 defined inboth members are aligned. A fastener 108 is then inserted through eachhole 108 and a nut 110 is meshingly engaged with each fastener 108.Positioning and securing second member 94 to first member 92 in theabove described manner results in (i) sealed cavity 100 being definedbetween first member 92 and second member 94, (ii) gasket 112 beingpositioned in contact with membrane 144 and located over sealant 148,and (iii) a portion 166 of chromatographic bed 16 being located withinsealed cavity 100.

One end of a metallic conduit 118 is then placed in fluid communicationwith an exit orifice 116 defined in second member 94 as shown in FIG. 4.The other end of conduit 118 is placed in fluid communication with apump 122. One end of another metallic conduit 120 is also placed influid communication with pump 122 while the other end of conduit 120 isplaced in fluid communication with an entrance orifice 114 defined insecond member 94. Furthermore, a coiled portion 138 of conduit 120 islocated within cooling unit 132. Coiled portion 138 is in contact with acooling fluid 136 contained within cooling unit 132.

Temperature sensor 126 is positioned within sealed cavity 100 such thattemperature sensor 126 can measure the temperature of a fluid beingadvanced through sealed cavity 100. Note that temperature sensor 126 canalso be positioned within conduit 118 adjacent to exit orifice 116.Temperature sensor 126 is electrically coupled to temperature controlunit 128 by an electrical line 130. Temperature control unit 128 iselectrically coupled to cooling unit 132 by electrical line 134. Itshould be understood that temperature sensor 126 detects the temperatureof a fluid being advanced through sealed cavity 100 and communicates thetemperature data to temperature control unit 128. Temperature controlunit 128 then controls the operation of cooling unit 132 based upon thedata received from temperature sensor 126. For example, temperaturecontrol unit 128 may cause cooling unit 132 to cool or warm the coolingliquid 136 within cooling unit 132 depending upon the data received fromtemperature sensor 126. Therefore, it should be appreciated thattemperature sensor 126, cooling unit 132, and temperature control unit128 cooperate in a well known manner to maintain any fluid beingadvanced through conduit 120, and therefore sealed cavity 100, within apredetermined temperature range.

Container 90, with plate 12 contained therein, is then positionedrelative to tank 73 so that end 20 of plate 12 is located below surface26 of mobile phase 24. However, sample area 22 is located within sealedcavity 100 above surface 26 of mobile phase 24. Cathode 30 and anode 28are electrically coupled to power source 40 via electrical wires 44 and42 as previously discussed. In addition, anode 28 is positioned incontact with mobile phase 24 as shown in FIG. 4. Moreover, cathode 30 ispositioned in contact with cathode portion 170 of plate 12 which islocated outside of sealed cavity 100 (see FIG. 7). In addition, wick 75is interposed between platinum foil 52 and chromatographic bed 16 aspreviously discussed. An electrical potential is then generated betweencathode 30 and anode 28 with power source 40 to develop plate 12 in amanner substantially identical to that described above in reference toarrangements 10 and 37.

Once plate 12 begins to develop, pump 122 is actuated so that a fluid168 is advanced under pressure into sealed cavity 100 in the directionsindicated by arrows 140 (see FIG. 4). Note that (i) baffles 124 attachedto an inside wall of sealed cavity 100 cause fluid 168 to travel throughsealed cavity in a serpentine fashion and (ii) membrane 144 preventsfluid 168 from coming into contact with chromatographic bed 16. Itshould be understood that fluid 168 is advanced into sealed cavity 100so that the pressure within sealed cavity 100 is greater than thepressure outside of sealed cavity 100. For example, pressure withinsealed cavity 100 can be in the range of about 3 to 50 atmospheres.Placing sealed cavity 100 under the aforementioned pressure alsosubjects the portion 166 of the chromatographic bed 16 located withinsealed cavity 100 under the same pressure. Having portion 166 of thechromatographic bed 16 under pressure during the development of plate 12is an important aspect of the present invention since it substantiallyenhances the separation efficiency of arrangement 88. Furthermore,advancing fluid 168 into sealed cavity 100 under pressure places fluid168 and plate 12 in a heat exchange relationship with chromatographicbed 16 such that temperature control unit 128, in cooperation withcooling unit 132, can maintain the temperature of plate 12 within apredetermined range. In particular, maintaining plate 12 within apredetermined temperature range prevents ohmic over heating of plate 12which can reduce the separation efficiency of arrangement 88.

The fluid 168 being advanced through sealed cavity 100 is preferably achemically inert, liquid which has a very low electrical conductivity.Such a liquid is commercially available from the 3M Corporation, locatedin Minneapolis, Minn., as Fluorinert or FC-77. In addition, megohmquality water could be used.

Once plate 12 develops, container 90 can be disassembled by removingfasteners 108 and separating first element 92 from second element 94.After separating first element 92 from second element 94 plate 12 can berecovered.

Referring now to FIG. 8, there is shown an arrangement 154 which issubstantially similar to arrangement 88 and therefore a detaileddescription of all the elements of arrangement 154 will not be providedherein. It should be understood that arrangement 154 functions insubstantially the same way, and has the same advantages, as discussedabove in reference to arrangement 88. In particular, it should beunderstood that arrangement 154 also drives the mobile phase 24 throughthe chromatographic bed 16 of plate 12 utilizing an electrical potentialgenerated between first electrode 28 and second electrode 30. However,the container 90 of arrangement 154 has a bladder 156 positioned withinsealed cavity 100 rather than a number of baffles 124 and membrane 144.

Bladder 156 defines a void 158. Additionally, bladder 156 has an exitaperture 160 defined therein which is in fluid communication withconduit 118. Bladder 156 also has an entrance aperture (not shown) whichis in fluid communication with conduit 120 (see FIG. 4). When developingplate 12 with arrangement 154, pump 122 advances fluid 168 underpressure into void 158 which causes (i) the pressure within sealedcavity 100 to be greater than the pressure outside of sealed cavity 100and (ii) bladder 156 to be urged into contact with chromatographic bed16 of plate 12. Urging bladder 156 into contact with chromatographic bed16 places chromatographic bed 16 under pressure so as to substantiallyincrease the separation efficiency of arrangement 154. Furthermore,urging bladder 156 against plate 12 places fluid 168 in void 158 in aheat exchange relationship with chromatographic bed 16 such thattemperature control unit 128, in cooperation with cooling unit 132, canmaintain the temperature of plate 12 within a predetermined range.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, such illustration and description isto be considered as exemplary and not restrictive in character, it beingunderstood that only the preferred embodiments have been shown anddescribed and that all changes and modifications that come within thespirit of the invention are desired to be protected. For example, whileplate 12 is shown being orientated in a vertical position herein otherorientations are contemplated, e.g. horizontal. Furthermore, the abovedescribed invention can be used with bi-directional chromatography inwhich the mobile phase is advanced in two opposite directions. Thisarrangement involves two parallel cathodes with an anode being centrallylocated relative to the cathodes. In addition, nonaqueous mobile phasescan be utilized with an appropriate chromatographic bed. Moreover, thecathode can be positioned within sealed cavity 100. Furthermore, itshould be understood that the present invention lends itself tosimultaneously separating multiple samples at a time on a single plate12. For example, 80 samples could be separated on a 40 cm wide plate 12.

What is claimed is:
 1. An arrangement for performing chromatography witha mobile phase and a chromatographic bed having a stationary phase,comprising: a support assembly configured to support saidchromatographic bed and said stationary phase, said support assemblyhaving a sealed cavity configured so that when said support assembly issupporting said stationary phase a portion of said chromatographic bedand said stationary phase extends into said sealed cavity; a firstelectrode positioned in contact with said mobile phase; and a secondelectrode positioned in contact with said stationary phase, whereincreating an electrical potential between said first electrode and saidsecond electrode causes said mobile phase to be advanced through saidchromatographic bed positioned within said sealed cavity.
 2. Thearrangement of claim 1, wherein: said first electrode includes an anode,and said second electrode includes a cathode.
 3. The arrangement ofclaim 2, wherein: said stationary phase includes a thin layerchromatography plate having a cathode portion and an anode portion, saidmobile phase includes a liquid, said anode portion of said thin layerchromatography plate is in contact with said liquid when said portion ofsaid stationary phase is located within said sealed cavity, said cathodeis in contact with said cathode portion of said thin layerchromatography plate, said anode is in contact with said liquid of saidmobile phase, and creating an electrical potential between said cathodeand said anode causes said liquid of said mobile phase to migratethrough said chromatographic bed in a direction away from said anodeportion and toward said cathode portion.
 4. The arrangement of claim 3,wherein: said cathode includes a platinum wire attached to a piece ofplatinum foil, and said platinum foil is positioned in contact with saidcathode portion of said thin layer chromatography plate.
 5. Thearrangement of claim 1, further comprising: a fluid positioned withinsaid sealed cavity of said support assembly so that pressure within saidsealed cavity is greater than pressure outside of said sealed cavity. 6.The arrangement of claim 1, further comprising: a bladder having a voiddefined therein, said bladder being positioned within said sealedcavity; and a fluid positioned within said void of said bladder so thatsaid bladder is urged toward said stationary phase positioned withinsaid sealed cavity.
 7. The arrangement of claim 6, wherein: said fluidpositioned within said void of said bladder is a liquid.
 8. Thearrangement of claim 7, further comprising: an absorbing elementpositioned in contact with said cathode portion of said thin layerchromatography plate so that said absorbing element absorbs said liquidwhen said electrical potential is created between said anode and saidcathode.
 9. The arrangement of claim 1, further comprising: a housingsecured to said support assembly, said housing having an interiorchamber configured to receive a cooling fluid, wherein said interiorchamber is located relative to said stationary phase so that saidcooling fluid is positioned in a heat exchange relationship with saidstationary phase when said electrical potential is created between saidanode and said cathode.
 10. An arrangement for performing chromatographywith a mobile phase and a chromatographic bed having a stationary phase,comprising: a support assembly configured to support said stationaryphase, said support assembly having a sealed cavity configured so thatwhen said support assembly is supporting said stationary phase a portionof said chromatographic bed and said stationary phase extends into saidsealed cavity; a fluid positioned within said sealed cavity so that (i)said fluid is placed in a heat exchange relationship with saidstationary phase and (ii) pressure within said sealed cavity is greaterthan pressure outside of said sealed cavity; an anode positioned incontact with said mobile phase; and a cathode positioned in contact withsaid stationary phase, wherein creating an electrical potential betweensaid anode and said cathode causes said mobile phase to be advancedthrough said chromatographic bed positioned within said sealed cavity.11. The arrangement of claim 10, further comprising: a pump in fluidcommunication with said sealed cavity, wherein said pump advances saidfluid through said sealed cavity via an entrance orifice defined in afirst wall of said sealed cavity and an exit orifice defined in a secondwall of said sealed cavity.
 12. The arrangement of claim 10, wherein:said stationary phase includes a thin layer chromatography plate havinga cathode portion and an anode portion, said mobile phase includes aliquid, said anode portion of said thin layer chromatography plate is incontact with said liquid when said portion of said stationary phase islocated within said sealed cavity, said cathode is in contact with saidcathode portion of said thin layer chromatography plate, said anode isin contact with said liquid of said mobile phase, and creating anelectrical potential between said cathode and said anode causes saidliquid of said mobile phase to migrate through said chromatographic bedin a direction away from said anode portion and toward said cathodeportion.
 13. The arrangement of claim 10, further comprising: anabsorbing element positioned in contact with said stationary phase sothat said absorbing element absorbs said mobile phase when saidelectrical potential is created between said anode and said cathode.